Abstract Background Tacrolimus, cyclosporine, and sirolimus are commonly used immunosuppressive drugs with narrow therapeutic windows requiring consistent monitoring to avoid drug toxicity. The Abbott Architect i2000 tacrolimus, sirolimus, and cyclosporine assays each require a pre-analytical whole blood lysing extraction step prior to analysis on the i2000. This extraction is largely manual, requiring the combination of an extraction reagent with the patient sample, followed by an incubation and centrifugation/pelleting steps, after which, the resulting supernatant is assayed for drug concentration. Each assay has its own unique extraction reagent and incubation steps, albeit all three procedures are quite similar. A clinician-requested investigation into a questionably elevated tacrolimus level led us to investigate the effect of substituting extraction reagents and procedures between the three assays. In this study, we investigated the effect of substituting extraction procedures and reagents on assayed immunosuppressive drug values between the Abbott tacrolimus, sirolimus, and cyclosporine immunoassays. Methods Whole blood was obtained from three patients who had previously had either their tacrolimus, sirolimus, or cyclosporine level assayed within the normal therapeutic range. For each patient, the cyclosporine, tacrolimus, or sirolimus level was re-assayed in triplicate, with the substitution of the other two assay’s extraction procedures performed prior to analysis. Such as: tacrolimus extraction was performed, then assayed for sirolimus or cyclosporine. This procedure was repeated for all three drugs and extraction procedures, affording 9 trials in total. The procedure was repeated with three samples obtained from patients with no measurable immunosuppressive drug levels. Additionally, cross-reactivity in the assay extraction reagents was assessed by measuring, in triplicate, immunosuppressive drug concentration on each extraction reagent without the addition of patient whole blood. Results Analysis in patients undergoing immunosuppressive drug therapy afforded the following changes in assayed drug concentration: Tacrolimus substituting sirolimus, cyclosporine extractions, 5.60 to 8.13, 8.47 ng/mL; sirolimus substituting cyclosporine, tacrolimus extractions 13.63 to 4.60, 8.07 ng/mL; cyclosporine substituting sirolimus, tacrolimus extractions 274.6 to 391.3, 757.30 ng/mL. Analysis in patients with no measurable drug content resulted in no change in assayed drug concentration with exception of tacrolimus, in which tacrolimus substituting sirolimus, cyclosporine extractions afforded <1.50 to 4.40, 4.00 ng/mL. Cross-reactivity trials yielded the following drug concentrations: tacrolimus precipitant: tacrolimus: >30.00, cyclosporine: >1500, sirolimus: 5.97 ng/mL; cyclosporine precipitant: cyclosporine: >1500, tacrolimus: 7.13, sirolimus 1.53 ng/mL; sirolimus precipitant: sirolimus: 5.00, tacrolimus: 8.67, cyclosporine: <25.00 ng/mL. Conclusions The use of incorrect extraction reagents and procedures resulted in both increased and decreased assayed immunosuppressive drug concentrations. These changes in drug concentration were observed in both samples containing, and not containing, immunosuppressive drugs. Significant cross-reactivity was observed in all three assay extraction reagents resulting inappropriately elevated assayed drug concentrations in all three assays. This highlights the need for comprehensive technologist training and effective pre-analytical workflows to prevent errors, such as reagent substitution, from occurring. This study has clinical implications into investigations of spurious immunosuppressive drug values, and substitution of assay extraction reagents and procedures should be investigated when performing root cause analysis into incorrectly reported immunosuppressive drug concentrations.